Developing Yeast Strains for Biomass-to-Ethanol Production

By Ronald Hector, Stephen Hughes and Xin Liang-Li
Although grain supplies will likely meet the immediate short-term needs for ethanol production, expansion beyond this to meet more ambitious targets will require alternative feedstocks. Lignocellulosic biomass from agricultural residues, municipal paper waste, dedicated energy crops and multiple other sources is projected to be a major renewable feedstock for sustainable production of biofuels.

The conversion of lignocellulose to ethanol involves a series of enzymatic steps for hydrolysis or saccharification of the constituent polysaccharides, and subsequent fermentation of the released hexose and pentose sugars. Additionally, a pretreatment step is required to disrupt the tightly packed cellulose structure and allow access to the enzymes. Many popular pretreatment conditions are not mild, and the yeast and enzymes must survive the chemicals used in the process.

According to Joseph Rich, leader of the USDA Bioproducts and Biocatalysis Research Unit in Peoria, Ill., "Industry is awaiting the microorganism that can produce high levels of ethanol in large-scale fermentation containing the hydrolysate consisting of both pentose and hexose sugars released by mechanical, enzymatic and chemical treatment of lignocellulosic feedstocks."

A variety of processes show potential for cellulose conversion into ethanol.
SOURCE: USDA Agricultural Research Service

Streamlining the Process
Many of the enzymes proposed for use in separate hydrolysis and fermentation processes are inhibited by their products, necessitating the addition of large quantities of enzyme to reach significant monosaccharide concentrations prior to fermentation. Anticipated costs of enzymes and pretreatment make the process of converting biomass to ethanol more expensive than the presently used and well-established starch-based processes. These costs are partially offset by the use of less expensive and abundant sources of lignocellulose from trees, shrubs, switchgrass or agricultural crop residues. Further economic advantages might be attained through more streamlined processes such as simultaneous saccharification and fermentation and consolidated bioprocessing.

The simultaneous saccharification and fermentation process combines polysaccharide hydrolysis and fermentation in one step, but still relies on the addition of exogenously produced enzymes. The simultaneous saccharification and fermentation that occurs in this type of process is an attractive method for keeping monomeric sugars at low enough concentrations to avoid enzyme inhibition, thus reducing costs by decreasing the amount of enzyme needed for the process.

The consolidated bioprocessing process takes streamlining a step further and combines the production of enzymes with the same organism used to ferment the released sugars to ethanol, all occurring in a single reactor. "To achieve the DOE 30x'30 plan goal of 60 billion gallons of biofuels, [or] 30 percent of the motor gasoline supply by 2030, will certainly require advancements such as the one-step bioprocessing with Saccharomyces cerevisiae," according to Seth Snyder at Argonne National Laboratory.

Lee Lynd of Dartmouth College and Willem van Zyl of the University of Stellenbosch have succeeded in expressing cellulases in S. cerevisiae. As a promising first step toward a consolidated bioprocessing process, the recombinant yeast strain they generated was able to produce some ethanol from cellulose without added enzymes.

Pentose Fermentation
Organisms that can ferment pentose sugars like xylose and arabinose, in addition to glucose, are essential for an economical process. Hemicellulose, which accounts for approximately 25 percent to 40 percent of lignocellulose, is mainly composed of xylose. While S. cerevisiae is good at converting glucose to ethanol, it does not have the metabolic capacity to utilize xylose.

Many years of research have been applied to engineer a yeast strain that can metabolize xylose as well as the hexose sugars found in biomass. Much of this research has recently focused on enhancing the fermentation performance of S. cerevisiae strains expressing heterologous enzymes from bacterial or fungal xylose utilization pathways. Research labs around the world have been trying to solve the problem of poor xylose utilization. Identifying the limiting metabolic steps that block efficient conversion of xylose to ethanol in these strains has been one of the goals for Thomas Jeffries at the USDA Forest Service's Forest Products Laboratory in Madison, Wis.

Various xylose fermenting yeast strains have been produced and some have found industrial application for processes using lignocellulosic feedstocks. Jack Pronk and his group at Delft University of Technology recently achieved rapid anaerobic fermentation of xylose and arabinose by engineered S. cerevisiae strains that express heterologous, pentose-isomerase based pathways. "Now that the hurdle of efficient pentose fermentation by yeast is being overcome, functional expression of hydrolyzing enzymes in the yeast is an important next challenge for yeast metabolic engineering," Pronk says.

Xylose fermenting yeast strains that express the fungal xylose pathway genes have also been improved upon and are being used in industrial processes, such as the yeast strain from Purdue University's Nancy Ho and strains being developed at Lund University by Bärbel Hahn-Hägerdal.

Yeast strains developed at the National Center for Agricultural Utilization Research have been engineered for enhanced pentose utilization by adding metabolic correction genes in addition to the genes required for growth on xylose. These genes were obtained from collaborations with Josh LaBaer, director of the Harvard Institute of Proteomics. To further improve these ethanologenic yeast strains for industrial use, scientists at NCAUR are also engineering yeast to express proteins that increase uptake of pentose sugars.

A depiction of xylose metabolic pathways
SOURCE: USDA Agricultural Research Service

Lignocellulose model showing lignin, cellulose and hemicellulose
SOURCE: USDA Agricultural Research Service

Enzyme Requirements for Lignocellulosic Feedstocks
Although S. cerevisiae is a proven industrial ethanol producer in traditional starch-based processes, it will be no easy task to provide this microorganism with the ability to convert lignocellulosic biomass to ethanol. The carbohydrate components of lignocellulose (cellulose and hemicellulose) are tightly bound to lignin, making the sugars largely inaccessible to enzymes. "Before enzymatic hydrolysis, pretreatment with acid or alkali is generally needed to fully maximize the release of sugars from any lignocellulosic biomass," says Badal

Saha at the NCAUR Fermentation Biotechnology Research Unit.
For consolidated bioprocessing, S. cerevisiae must not only ferment both hexoses and pentoses under industrial conditions with high ethanol yield and productivity, it must also express and produce enzymes at sufficient levels to maintain hydrolysis and fermentation of biomass to ethanol. Enzymatic conversion of cellulose to sugars that yeasts can

Ferment requires the concerted action of three types of cellulase. Due to the heterogeneity and complexity of hemicellulose, its conversion requires an even larger list of enzymes.

For robust and complete conversion of polysaccharides locked in biomass, the ultimate ethanologens will need to produce at least a dozen enzymes of different catalytic activities.

Developing New Biocatalysts
Producing a yeast strain with optimized sets of cellulases and hemicellulases requires screening thousands of combinations of these biomass-degrading enzymes for enzyme activity. Automation is essential in carrying out these operations. A team of scientists at the NCAUR laboratory has been successful in designing a robotic platform and creating the automated molecular biology routines necessary to screen for the most effective set of enzymes.

The genes for these enzymes may exist in organisms contained in the ARS culture collection and from organisms isolated from environments such as cattle rumen, hot springs, termite guts and ocean thermal vents. Sookie Bang at the Center for Bioprocessing Research and Development located at the South Dakota School of Mines and Technology is isolating extremeophiles from the National Science Foundation-sponsored Deep Underground Science and Engineering Laboratory as a source of novel enzymes that have been selected for more than 125 years at temperatures in excess of 140 degrees Fahrenheit in the harsh deep-mine conditions. These enzymes hold great promise for use in producing lignocellulose-degrading yeast strains.

Assuming appropriate enzymes are identified, a critical question remains. Is S. cerevisiae capable of simultaneously expressing the genes for all the different enzymes necessary to hydrolyze cellulose and hemicellulose as well as ferment pentose sugars? Indications are increasing that ethanol production by an ethanologen that has the ability to efficiently hydrolyze pretreated biomass and metabolize the resulting sugars is feasible. However, says Michael Cotta, leader of the Fermentation Biotechnology Research Unit at NCAUR, "Considerable research and development are still needed to develop the optimum enzymes, organisms and processes that will be able to generate a sustainable biomass to ethanol process."

Ronald Hector, Stephen Hughes and Xin Liang-Li are research molecular biologists with the USDA's Agricultural Research Service. Reach Hector at or (309) 681-6098. Reach Hughes at or (309) 681-6176. Reach Liang-Li at or (309) 681-6327.

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